3M™ Petrifilm™ Rapid Yeast and Mold Count Plate for the Enumeration of Yeasts and Molds in Dried Cannabis Flower: AOAC Official MethodSM 2014.05

Abstract Background The 3MTM Petrifilm™ Rapid Yeast and Mold Count (RYM) Plate is a sample-ready culture medium system which contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration. Objective The 3M Petrifilm RYM Plate was validated for the enumeration of yeast and mold in dried cannabis flower through the AOAC Emergency Response Validation process. Methods The performance of the 3M Petrifilm RYM Plate was compared to dichloran rose bengal chloramphenicol (DRBC) agar. Matrix data were normalized by log10 transformation and performance indicators included repeatability, difference of means, and inclusivity/exclusivity. Results These studies demonstrated the 3M Petrifilm RYM Plate method detects and enumerates yeasts and molds from dried cannabis flower at low, medium, and high contamination levels. The average log counts at 25 or 28°C for 60 to 72 h were equivalent to the average log counts of the DRBC reference method at low, medium and high levels. In strain studies, all 71 yeasts and molds tested produced typical colony morphology on 3M Petrifilm RYM Plates. Of the 32 non-target bacterial strains tested, none were detected on 3M Petrifilm RYM Plates. Conclusion The 3M Petrifilm RYM Plate is a reliable method for the enumeration of live yeast and mold in dried cannabis flower. Highlights The 3M Petrifilm RYM Plate allows for rapid detection of yeast and mold within 60 to 72 h of incubation. Up to 40 sample-ready plates can be stacked during incubation to save space.

mycotoxins, a by-product that is toxic to humans and animals. Several yeast and mold have been found to be prevalent in cannabis, including Cryptococcus, Mucor, Aspergillus fumigatus, A. niger, and A. flavus (1,2). Aspergillus species niger, flavus, and fumigatus are known for aflatoxin production, a type of dangerous mycotoxin that can be lethal (3). For this reason, regulations exist to limit the allowable TYMC counts for the purposes of protecting consumer safety (4).
The 3M TM Petrifilm TM Rapid Yeast and Mold Count (RYM) Plate is a sample-ready culture medium system which contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration.
The performance of the 3M Petrifilm RYM Plate method was previously shown to be comparable to ISO 21527:2008 parts 1 and 2, and the U.S. Food and Drug Administration Bacteriological Analytical Manual Ch. 18 (FDA-BAM Ch. 18) reference methods (5-7) for enumeration of yeast and mold in food and environmental surfaces at 25 6 1 C and 28 6 1 C after 48 to 60 h (highwater activity matrixes: yogurt, frozen bread dough, fermented salami, sour cream, ready-made pie, raw frozen ground beef patties (77% lean), ready-to-eat deli sandwiches, sliced apples; low-water activity matrixes: raw almonds, dehydrated soup; and environmental surfaces: stainless steel, sealed concrete, and rubber (8)(9)(10)). The method has Final Action status as Official Methods of Analysis SM (OMA) 2014.05 (11). The current matrix extension study compares the performance of the 3M Petrifilm RYM to dichloran rose bengal chloramphenicol (DRBC) agar for the enumeration of yeast and mold in dried cannabis flower (9-tetrahydrocannabinol [THC] >0.3%) at 60 to 72 h.

Matrix Extension Validation Study
This validation study was conducted as an Emergency Response Validation (ERV) process within the AOAC Research Institute (RI) Performance Tested Method SM (PTM) program. The validation followed the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces (12) and Standard Method Performance Requirements (SMPR) for Viable Yeast and Mold Count Enumeration in Cannabis and Cannabis Products (13), which was developed by the AOAC Cannabis Analytical Science Program. Inclusivity and exclusivity testing was conducted by 3M (St. Paul, MN 55144-1000, USA), ADRIA Dé veloppement (ZA Creac'h Gwen F-29196 QUIMPER Cedex, France), and Q Laboratories (Cincinnati, OH, USA). Cannabis flower matrix study materials were prepared by Steadfast Analytical Laboratory (Hazel Park, MI, USA). Test portions were blindcoded and provided to North Coast Testing Laboratories of Michigan, (Adrian, MI, USA) for analysis using the 3M Petrifilm RYM Plate. Additional cannabis flower matrix study materials were prepared, blind-coded, and analyzed by Aurum Laboratories (Durango, CO, USA).

Inclusivity/Exclusivity Study
An inclusivity/exclusivity study of the 3M Petrifilm RYM Plate was previously performed using 42 strains of yeast and mold and 4 strains of non-target organisms as part of the AOAC PTM 121301 and AOAC OMA 2014.05 validation studies (8,9). Evaluation of target and non-target strains was not required for yeast and mold methods per AOAC Appendix J (11) when AOAC PTM 121301 and OMA 2014.05 studies were conducted. A select number of target and non-target organisms were included in the validation studies as supplemental data on the performance of the method. For the organisms obtained from Microbiologics, Inc. as LYFO DISK V R preparations, one pellet was added to 10 mL pre-warmed (37 C) 0.1% peptone water (PW) and then mixed on a vortex mixer until the pellet was completely dissolved. The pellet suspension was serially diluted as needed (the pellet counts provided by the manufacturer served as guidance in establishing the dilution scheme). The 3M Petrifilm RYM Plates were then incubated at 25 and 28 C and enumerated at 48 and 60 h.
The ERV protocol required data for specific inclusivity and exclusivity organisms commonly found in cannabis matrixes. Additional strains to satisfy this requirement were provided using a subset of data from ADRIA Dé veloppement for a previously conducted NF Validation by AFNOR certification (3M 01/13-07/ 14) in comparison to ISO 21527 part 1 and part 2 (14 inclusivity, 20 exclusivity), or were tested by Q Laboratories (17 inclusivity, 8 exclusivity).
In the study conducted by ADRIA Dé veloppement, the inclusivity strains were grown in Sabouraud broth at 25 C. The nontarget strains were grown in appropriate media at appropriate temperatures. All organisms were tested at two incubation temperatures, 25 6 1 C and 28 6 1 C. Plates were examined and results recorded at 48 h (data not published in NF Validation by AFNOR certification) and 60 and 72 h (10).
In the study conducted by Q Laboratories, yeast organisms were propagated from a stock culture stored at À70 C to potato dextrose broth and incubated at temperatures optimal for growth. Following incubation, yeast organisms were diluted to 100Â the LOD of the 3M Petrifilm RYM Plates. Mold organisms were propagated from a stock culture stored at À70 C to Sabouraud dextrose agar and incubated for 5-7 days at 30 6 1 C. Following incubation, mold spores were harvested for inclusivity testing by washing cultures with Butterfield's phosphatebuffered dilution water. The mold wash was then diluted to 100Â the LOD of the 3M Petrifilm RYM Plates.
Exclusivity organisms were propagated from a stock culture stored at À70 C to trypticase soy agar with 5% sheep blood and incubated at conditions optimal for growth. Following incubation exclusivity organisms were transferred to the non-selective brain heart infusion broth and incubated at conditions optimal for growth. Exclusivity cultures were analyzed undiluted.
All organisms were randomized in a blind-coded study and plated onto 3M Petrifilm RYM Plates as indicated in the instructions for use. All organisms were tested at two incubation temperatures, 25 6 1 C and 28 6 1 C. Plates were examined and results recorded at 48 and 60 h Colonies were determined to be positive or negative based on the product instructions.

Matrix Study
Cannabis test materials were prepared by Steadfast Analytical from an inventory of retained samples from its Michiganlicensed grower, patient, and caregiver customers. Steadfast combined cannabis samples to produce batch materials targeting at least 1000 g at a low level [<1000 colony-forming units (cfu)/g), a medium level (1000-10 000 cfu/g), and a high level (10 000-100 000 cfu/g). Batches were manually mixed in an aseptic manner until homogeneous. For each contamination level, five replicate test portions (10 g) were quantified by spread plating aliquots of diluted test portions onto DRBC agar plates. Table 1 summarizes the average cfu/g of yeast and mold for each contamination level that was provided to laboratories for analysis in the study.
Individual 10 g test portions from each contamination level were placed in sterile filter Whirl-Pak bags. Five bagged test portions from each of the three contamination levels were selected for each candidate method participating in the ERV project. Test portions were assigned an identification tag in Michigan's Marijuana Regulatory Agency seed-to-sale system for distribution and tracking. This served to blind-code the contamination level of the test portions. The test portions were also assigned random sample numbers for reporting results to AOAC.
Personnel from each of the participating independent laboratories were responsible for picking up and transporting the test portions to their laboratories on Monday, December 7, 2020. Participating laboratories were instructed to analyze samples on Tuesday, December 8, 2020, following the user guides provided with the candidate methods. In addition to the candidate methods, all test portions were enumerated using DRBC agar as described in the DRBC reference section. For the 3M Petrifilm RYM Plate method, North Coast Testing Laboratories of Michigan conducted the matrix evaluation.
A second set of cannabis samples targeting a low level (approximately 1000 cfu/g) and a high level (approximately 100 000 cfu/g) were prepared by Aurum Laboratories. A 50-60 g sample of cannabis flower matrix was made by combining previously tested sample inventory. Cannabis flower samples that previously tested at/or slightly above 1000 cfu/g were combined into one large sample. Cannabis flower samples that previously tested >74 000 cfu/g were combined into one large sample. The combined samples were homogenized using a sterile stainless steel mortar and pestle. A portion of the homogenized sample was used for cannabinoid analysis to confirm THC >0.3%. From each homogenized sample, five 10 g samples were aseptically weighed out into sterile Whirl-Pak filter bags. Samples were blind-coded prior to plating at Aurum Laboratories.

Candidate Method
All analyses at both laboratories were performed using paired test portions. Test portions were prepared for analysis as described in the 3M Petrifilm RYM Plate method. Ten gram portions were homogenized in 90 mL 0.1% PW. Tenfold dilutions were made by transferring 10 mL into 90 mL PW and shaking 25 times in a 1-foot arc within 7 s to ensure homogeneity. A 1 mL aliquot from each dilution (10 À1 , 10 À2 , 10 À3 , and 10 À4 ) was plated in duplicate on Petrifilm RYM Plates. Two sets of plates were prepared, one set incubated at 25 6 1 C and the second set incubated at 28 6 1 C. Plates were read and colonies recorded at 72 h (North Coast Testing Laboratories) or 60 h and 72 h (Aurum Laboratories). Plates containing counts between 10-150 were used to determine the final results. If mainly yeast are present, plates with 150 colonies are usually countable. When substantial amounts of mold were present and a more accurate count was obtained on the next dilution, the upper countable limit was lowered per the guidance in the 3M Petrifilm RYM Plate method and FDA-BAM Ch. 18. The counts from duplicate plates were averaged and used for the statistical analysis.

Reference Method
Paired test portions, prepared following the candidate method dilution protocol, were confirmed by spread plating aliquots of each dilution onto DRBC agar plates. From the initial dilution of sample (10 g homogenized in 90 mL PW), 1.0 mL was spread plated across two DRBC agar plates (0.5 mL on each plate) in triplicate (six total DRBC agar plates) or across three DRBC agar plates (0.33 mL on each plate) in triplicate (nine total DRBC agar plates) at North Coast Laboratory or Aurum Laboratories, respectively. Additionally, 0.1 mL of the 10 À1 , 10 À2 , and 10 À3 dilutions was plated in triplicate on DRBC to obtain the 10 À2 , 10 À3 , and 10 À4 dilutions respectively. The agar plates were allowed to dry and were then incubated at 25 6 1 C for 5-7 days before enumeration. Mold appeared as flat or fuzzy, spreading colonies with the natural pigmentation of the sporing structures, and yeast appeared as pink, smooth, raised colonies on DRBC agar plates (5). Plates containing counts between 10-150 colonies were enumerated as described in the FDA-BAM Ch. 18. The counts from triplicate plates were averaged and used for the statistical analysis. [Applicable to the enumeration of yeast and mold in the following high-water activity matrixes: yogurt, frozen bread dough, fermented salami, sour cream, ready-made pie, raw frozen ground beef patties (77% lean), ready-to-eat deli sandwiches, sliced apples; the following low-water activity matrixes: raw almonds, dehydrated soup, dried cannabis flower (THC > 0.3%); and the following environmental surfaces: stainless steel, sealed concrete, and rubber.] Caution: After use, the diluents and 3M Petrifilm RYM Plates may contain microorganisms that may be a potential biohazard as several foodborne molds have the ability to produce toxic metabolites known as mycotoxins. If further identification of a mold species is required, appropriate personal protective equipment (PPE) should be used when top film is retracted and exposure to spores or mycotoxins may occur. When testing is complete, follow current industry standards for the disposal of contaminated waste. Consult the material safety data sheet for additional information and local regulations for disposal. For information on potential biohazards, reference Biosafety in Microbiological and Biomedical Laboratories, 6th Ed., Section VIII-B: Fungal Agents.
The 3M Petrifilm RYM Plates contain chloramphenicol and chlortetracycline, potent broad spectrum antibiotic drugs commonly used in yeast and mold enumeration. The drugs, when used in humans, is associated with many toxic effects. Care should be taken to avoid coming into direct contact with the gel on the plates.
See Tables 2014.05A and 2014.05B for a summary of results of the collaborative study. The result for each collaborating

A. Principle
The 3M Petrifilm RYM Plate is a sample-ready culture medium system, which contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration. 3M Petrifilm RYM Plates are used for the enumeration of yeast and mold in as little as 48 h in the food and beverage industries and 60 to 72 h in the cannabis industry. 3M Food Safety is certified to International Organization for Standardization (ISO) 9001 for design and manufacturing.   Do not count colonies on the foam dam since they are removed from the nutrient medium. (j) Yeast colonies appear raised and small with defined edges.

D. Sample Preparation
Colonies may appear pink/tan or blue/green in color. Estimation can only be done by counting the number of colonies in one or more representative squares and determining the average number per square. The average number can be multiplied by 30 to determine the estimated count per plate. If a more accurate count is required, the sample will need to be retested at higher dilutions. When the sample contains substantial amounts of mold, depending on the type of mold, the upper countable limit may be at user discretion.

Results
(a) Inclusivity/Exclusivity.-Results from the inclusivity and exclusivity testing conducted at Q Laboratories have been combined with the results from ADRIA Dé veloppement and the original 3M Petrifilm RYM Plate AOAC validation and are presented in Table 2 (inclusivity) and Table 3 (exclusivity). Seventy-one yeasts and molds tested showed typical colony morphology and thus were considered "positive" on 3M Petrifilm RYM Plates. Sixty-three out of 71 strains grew at both temperatures, 25 6 1 C and 28 6 1 C, and at both time points, 48 and 60 h. Two strains were not visible at 25 6 1 C at 48 h but showed growth at 25 6 1 C at 60 h and at 48 and 60 h at 28 6 1 C. Five strains were not visible at 48 h at 25 6 1 C or 28 6 1 C but showed growth at 60 h at both temperatures. One strain was not visible at 48 h at 25 6 1 C or 28 6 1 C, but showed growth at 72 h at 25 6 1 C and 60 h at 28 6 1 C. None of the exclusivity strains The 90 and 95% confidence intervals indicated there were no significant differences in detection or enumeration between the 3M Petrifilm RYM Count Plate method and the DRBC agar at the medium and high contamination levels at both temperatures, 25 6 1 C and 28 6 1 C, at 72 h. The 90% confidence interval was just outside the acceptance criterion for statistical equivalence at the low level at both temperatures, 25 6 1 C and 28 6 1 C at 72 h (À0.577, À0.236 and À0.682, À0.322, respectively). A higher standard deviation was observed on the DRBC agar plates (repeatability standard devidation (s r ) >0.2), which can lead to wider confidence intervals.
In order to investigate the recovery differences between the 3M Petrifilm RYM Count Plate method and DRBC at low levels further, a second study was conducted at low and high levels at both temperatures, 25 6 1 C and 28 6 1 C, at 60 and 72 h. In this study, the 90 and 95% confidence intervals of the bias between the two methods fell between À0.5 to 0.5 log 10 for each concentration indicating equivalence between the 3M Petrifilm RYM Count Plate method and the DRBC agar at the low and high contamination levels at 25 6 1 C and 28 6 1 C, at 60 or 72 h per AOAC SMPR 2021.009 recommended acceptance criteria.
During the second study, the laboratory noted that the DRBC reference plates appeared to have breakthrough growth of Pseudomonas. These bacterial colonies were initially mistaken as yeast colonies, which inflated the DRBC counts. DRBC reference plates were recounted with the omission of Pseudomonas. To confirm that the omitted colonies were bacteria, three visually distinct colonies were sent for genetic sequencing and identified as P. aeruginosa, P. juntendi, and P. lactis. In the first study, it was noted that the amount of contamination at the low level on DRBC (5360 cfu/g) was higher than expected based on the screening data provided by Steadfast (300 cfu/g Steadfast) while the recovery on 3M Petrifilm RYM Plate (325 and 318 cfu/g at 25 6 1 C and 28 6 1 C respectively) was aligned with the value provided by Steadfast for the low contamination level. It is

Conclusions
These studies have demonstrated that the 3M Petrifilm RYM Count Plate method is an accurate, specific, and repeatable method that detects and enumerates yeast and mold at 60 to 72 h from dried cannabis flower. It is recommended that the 3M Petrifilm RYM Plate, AOAC OMA 2014.05, be granted a matrix   extension for the detection and enumeration of yeasts and molds in dried cannabis flower.

Conflict of Interest
3M Company is the method developer. All authors from 3M Company are salaried employees of the company. North Coast Testing Laboratories, Aurum Laboratories, and Q Laboratories were contracted as independent laboratories to conduct the study per AOAC guidelines and received payment from 3M Company.